Process of preparing omicron-carbamyl-d-serine



United States Patent PROCESS OF PREPARING O-CARBAMYL-D- SERINE Guy Hagemann, Vincennes, and Lucien Peuasse and Jean Teillon, Paris, France, assignors to Les Laboratories Francais de Chimiotherapie, Paris, France, a corporation of France No Drawing. Original application Aug. 16, 1955, Ser. No. 528,832. Divided and this application Feb. 24, 1958, Ser. No. 716,859.

Claims priority, application France Aug. 19, 1954 8 Claims. (Cl. 195-80) The present invention relates to a new and improved process of preparing O-carbamyl-D-serine.

The present application is a division of our co-pending application Serial No. 528,832, filed August 16, 1955, now US. Patent 2,885,433 and entitled O-Carbamyl-D- Serine and a Process of Making Same.

It is one object of the present invention to provide a process of preparing the new and valuable O-carbamyl-D' senne.

Another object of the present invention consists in providing a new fermentation process of preparing said O- carbamyl-D-serine by cultivating Streptomyces polychromogenus and the like microorganisms.

Other objects of the present invention and advantageous features thereof will become apparent as the description proceeds.

In principle, the present invention consists in providing the new O-carbamyl-D-serine of the following formula Said O-carbamyl-D-serine has proved to be therapeutically effective in animals and humans and seems to have a favorable effect on the defense mechanism of the organism against infections.

The new O-carbamyl-D-serine forms colorless needles.

Its melting point, on slow heating, is 226-234 C. (with decomposition); its instantaneous melting point is 238 C. (with foaming); its rotatory power [u] =-19.6 (concentration: 2% in N hydrochloric acid solution) or [a] =+2 (concentration: 1% in water). Its ultraviolet absorption above 200 ,u is negligible.

According to the present invention O-carbamyl-D- serine is prepared by microbiological synthesis by cultivating under artifical conditions on a suitable nutrient medium a microorganism of the order Actino-mycetales and the family Streptomycetaceae. Said microorganism shall be designated hereinafter and in the claims attached hereto as Stre'ptomyces chromogenus. After fermentation is completed, the insoluble parts of the culture, such as the mycelium, are separated and removed by filtration or centrifuging.

O-carbamylD-serine is extracted from the resulting clear filtrate which may previously be concentrated, by absorption to a sulfonic ion exchange agent in its acid form. Such ion exchange agents are known to the trade, for instance, as Amberlite IR 120, Permutite Q, or Dowex 50. For this purpose, the filtrate is passed through a tube filled with said ion exchange agent. The column is then washed with water. The adsorbed O-carbamyl-D-serine is eluted from the washed column by 2,938,836 Patented May 31, 1960 means of an aqueous solution of a weak volatile base, such as ammonia, pyridine, triethylamine, and others. The eluate is concentrated by evaporation. On cooling, 0- carbamyl-D-serine precipitates and is purified by recrystallization. On cooling, O-carbamyl-D-serine precipitates and is purified by recrystallization. Cultivation of said microorganism may be effected either in surface cultures on a suitable nutrient medium or in submerged cultures under sterile conditions whereby the culture is agitated and sterile air is blown into and through the culture. The procedure followed and the apparatus employed are the same as conventionally used in the manufacture of antibiotics. The cultivating conditions, such as temperature and duration, and the composition of the nutrient medium can vary within wide limits without departing from the scope of the present invention. The principal characteristic features of this invention are the selection of a specific microorganism and the extraction of O-carbamyl-D- serine from the fermentation broth by means of a sulfonic ion exchange resin in acid form from which the new compound can be eluted by means of a weak volatile base.

Streptomyces polychromogenus has not been described heretofore. It has been isolated from a sample of Venezuelan soil. Its cultures obtained by selective cultivation on an agar nutrient medium exhibit the following characteristic properties: The growth of a pure culture at a temperature of 28 C. on a culture medium containing 1.0% of anhydrous glucose, 0.05% of di-potassium phosphate, 0.05% of asparagine, and

2.0% of agar is quite abundant. The vegetative mycelium has a light brown to pale yellow color. The aerial mycelium is first white and then changes its colors to a bright greenish blue which, on aging, becomes pale green. The edge of the colony is of arbon'zed (dentritic) contour. The soluble, discrete pigment is of verypale yellowish brown color. The aerial hyphae are more or less branched and are divided into fragments of chains of ovoid spores. Their terminal or apical parts do not form spirals.

Streptomyces polychromogenus does not reduce nitrates. It coagulates and peptonizes milk. On a potato starch nutrient medium, it secretes amylases. On a medium which consists of 0.25% of ammonium sulfate,

0.25% of monopotassium phosphate,

0.5% dipotassium phosphate,

0.1% of magnesium sulfate heptahydrate, 0.0006% of copper sulfate pentahydrate, 0.0001% of ferrous sulfate heptahydrate, 0.0008% of manganous chloride tetrahydrate, 0.00015 of zinc sulfate heptahydrate, and 2.0% of agar,

Strptomyces polychromogenus grows readily in the presence of sucrose, glucose, fructose; and d-xy-lose, but less readily in the presence of d-galactose and rhamnose. Lactose, on the other hand, is not assimilated.

Streptomyces polychromogenus is sensitive to exposure to elevated temperature. Optimum growth is achieved at a temperature of 28 C. A temperature of 37 C. impairs its multiply-ing power. Such a temperature prevents production of an aerial mycelium.

, Said microorganism is also sensitive to acid pH-values. Acid culture media inhibit and retard its growth. For instance, it is possible to cultivate cultures of said microorganism on potatoes only if the potatoes are impregnated with a buffer solution of a pH 7.0.

In aculture medium'which consists of 1% of pure glucose,

0.5% of soybean peptone, 0.5% ofan aqueous heart extract, 0.5% of sodium chloride,

0.1% of calcium carbonate, and 2.0% of agar,

of its ability to synthesize O-carbamyl-D-serine. In submerged cultures said microorganism can secrete a pigment which, usually, is of brown color but may also be of olive-green or dark violet color.

The species Streptomyces polychromogenus is kept in the collection of species of microorganisms of the Socit V Francaise de la Penicilline of Romainville, Seine, France, under the reference No. T 4473. A culture of said species Stleptomyces polychromogenus has been deposited with the American Type Culture Collection in Washington, D.C., under the No. ETCC 12,595.

The nutritive requirements of Streptomyces 'p0lychromogenus are not yet known exactly. However, numerous tests carried out with different sources of carbon and nitrogen have shown that it is possible to use, in a nutrient medium suitable for submerged cultivation,

the following nutrients: Soybean meal, distillers solubles, V bakers yeast, enzymatic caseine hydrolysates, crude glucose, dextrin. V V Thefollowing examples serve to illustrate the present invention without, however, limiting the same thereto.

EXAMPLE '1 V The inoculum required for cultivation under suitable conditions of sterility and while aerating at a tempera ture at or near 2830 C. is either directly composed of a suspension of spores of, Streptomyces' polychromegenus or is cultivated on submerged cultures of said Streptomyces species by incubation at 28 C. for 48 hours, while agitated on a shaking device, in a nutrient medium consisting of 1% of glucose,

0.5 of dry malt extract,

0.5 of soybean meal,

0.5 of sodium chloride,

0.5% of dry corn steepliquor, and 0.1% of calcium carbonate.

The last mentioned inoculum is especially suitable for inoculating large scale fermentation tanks.

In a 500 1. fermentation tank, there are introduced 300 l. of a nutrient medium consisting of 2.6% of de-oiled soybean meal,

1.0% of dextrin,

1.0% of crude glucose,

0.3% of magnesium sulfate heptahydrate, and 02% of calcium carbonate.

. After sterilization of said nutrient medium "by heating to 120 C. for 40 minutes, the medium is inoculated with a culture of Streptomyces polychromogenus prepared as indicated hereinabove. The inoculation medium is then kept at a temperature of 28 C. for 50 hours whereby it is agitated and aerated under the conditions ordinarily used in the manufacture of antibiotics. .The resulting culture is then centrifugedand-the clear filtrate is worked up for the recovery and'isolation of O-carbamyl-D-serine as described hereinafter in Example 3.

EXAMPLE 2 shaken on a shaking device in a chamber at 28 C.

Fermentation is interrupted after 5 days. The culture is centrifuged as described in Example 1.

EXAMPLE 3 One liter of the clear centrifuged solution obtained on fermentation under the conditions described in Example l or 2 is passed drop by drop through a column of 300 g. of Amberlite IR 120 in acid form (moist weight). The diameter of said column is about mm. The column is then washed with 500 cc. of water and, thereafter, is eluted with 100 cc. of an aqueous solution containing 12% of pyridine.

The resulting eluate is concentrated by evaporation in a vacuum at 40 C. to a volume of about cc. During the last stages of said concentration the product is deposited in an amorphous and faintly colored form on the walls of the flask. At said moment, there are added, while stirring vigorously, 15 cc. of methanol. Precipitation is completed by keeping the mixture at 0 C. for

' 24 hours. The precipitate is filtered over a fritted glass filted and is washed with 25 cc. of cold 50% methanol. The precipitate is again dissolved in 20 cc. of warm water and treated with 0.5 g. of vegetable charcoal at C. for 15 minutes. After centrifuging anddecanting,

" an equal volume of methanol is added and the mixture is allowed to crystallize at 0 C. for 24 hours. The resulting crystals are again recrystallized in the same manner and are obtained thereby in small colorless needles which are filtered off, washed with .methanol, and dried in a vacuum. 1 g. of O-carbamyl-D-serine is obtained. Its melting point (on the Maquenne block) is 238 C. (with foaming).

Analysis.C O N H =molecular Weight: 148. Calculated: 32.43% C; 5.44% H; 18.92% N. Found: 32.4% C; 5.5% H; 18.8% N.

EXAMPLE 4 Formation of the mono-dinitro phenyl derivative 0 O-carbamyl-D-serine mg. of O-carbamyl-D-serine and 200 mg. of sodium carbonate are dissolved in' 10 cc. of water. A solution of mg. of 2,4-dinitro fiuorbenzene in 50 cc. of ethanol is added thereto. The mixture is allowed to react at room temperature for 2 hours while agitating from time to time. The alcohol is evaporated in a-vacuum. The remaining aqueous solution is washed with ether and is then acidified by the addition of a few drops of concentrated hydrochloric acid. N-2,4-dinitro phenyl-O- carbamyl-D-serine is precipitated. On recrystallization of said compound in aqueous ethanol, needles are obtained which melt (on'the Maquenne block) at 166 C.

Analysis.--C H O N =molecular weight: 314. Calculated: 38.2% C; 3,2%;H; 17.8% N. Found: 38.0% C; 3.4% H; 17.6% N.

20 mg. of said compound are hydrolyzed by means of 3 cc. of 6 N hydrochloric acid in a sealed tubeat 100 C. for 4 hours. The resulting hydrolysateis extracted three times with ether. The yellow ethereal extractis evaporated to dryness. The resulting residue is subjected to chromatography and is found to be identical with synthetically produced dinitro phenyl serine.

It is, of course, understood that in place of- Streptomyces polychromogenus used in the preceding examples, there may be employedother microorganisms of the Streptomyces genus and especially mutants obtainedfrom Streptomyces polychromogenus and the like organisms by the action of mutating agents, such as X-ray radiation, ultraviolet radiation, treatment with nitrogen mustard gas and others.

The nutrient medium may contain other sources of carbon than those mentioned hereinbefore such as saccharose, glycerol, levulose, maltose. Said nutrients can be added in pure form or in the form of suitable concentrates. The amounts to be added may vary considerably. Amounts between about 0.5% and about 5% of the nutrient medium have proved satisfactory.

Other proteins or other nitrogen containing substances than those mentioned in the preceding examples may also be used as sources of nitrogen. Such substances are, for instance, fish meal, wheat-steeping liquor, whey, hydrolyzed gluten, linseed meal, peanut meal, cottonseed meal, lactalbumin, autol' zed yeast, hydrolyzed casein, meat peptones.

These proteins can also be added in pure form or, preferably, in their crude non-purified form as they are obtained as by-product of various industrial processes. The amounts to be added may also vary considerably and be between about 1% and about 4% of the nutrient medium.

Other butter substances than those mentioned in the preceding examples may be added to the nutrient medium in order to maintain the pH-value of the nutrient medium between about 6.0 and about 8.5, such as potassium mono-H-orthophosphate, sodium citrate, sodium acetate.

The temperature at which the cultures are incubated is between about 20 C. and about 32 C. Optimum results are obtained at about 28 C. The most favorable temperature is, of course, dependent upon the composition of the nutrient medium and the specific strain of microorganism used.

Elution of the adsorbed O-carbamyl-D-serine from the sulfonic ion exchange resin in acid form can be efiected by other eluting agents than those mentioned in the preceding examples, such as, for instance, 5% ammonia solution, 3% aqueous triethylamine solution, 3% aqueous tributylarnine solution.

Precipitation of the O-carbamyi-D-serine dissolved in water can be effected not only by methanol as disclosed in the preceding examples but also by other organic solvents which are miscible with water, such as ethanol, isopropanol, acetone.

As stated hereinbefore, O-carbamyl-D-serine has proved to be therapeutically eiiective.

O-carbamyl-D-serines synergistic efiect with antibiotics is especially interesting. For example, the combination of O-carbamyl-D-serine and chloramphenicol leads to very interesting results in the treatment of diseases caused by Staphylococcus aureus, Eberthella typhi, and Escherichia coli.

We claim:

1. In a process of producing O-carbamyLD-sefine, the

steps comprising cultivating Streptomyces polychromegenus in an aqueous, nutrient medium containing proteins, carbohydrates, and mineral salts and having a pH between about 6.0 and about 8.5 under aerobic conditions and with agitation at a temperature between about 20 C. and about 32 C. until formation of O-carbamyl-D- serine is completed, and recovering the resulting O-carbamyl-D-serine from the nutrient medium by adsorption to a sulfonic ion exchange resin in acid form and subsequent elution by means of an aqueous solution of a volatile weak base selected from the group consisting of pyridine and ammonia.

2. In a process of producing O-carbamyI-D-serine, the steps comprising inoculating a sterile aqueous nutrient medium of a pH between about 6.0 and about 8.5, said nutrient medium containing proteins, carbohydrates, and mineral salts, with Streptomyces polychromogenus, incubating the resulting mixture under aerobic conditions at a temperature between about 20 C. and about 32 C., separating solid matter from the fermentation broth, passing said fermentation broth through a sulfonic ion exchange resin in acid form, washing said ion exchange resin, eluting therefrom the adsorbed O-carbamyl-D- serine by means of an aqueous solution of a volatile weak base selected from the group consisting of pyridine and ammonia, concentrating the resulting eluate, and recovering therefrom the precipitated O carbamyl-D- serine.

3. A process according to claim 1, wherein fermentation is effected in submerged culture.

4. A process according to claim 1, wherein fermentation is efiected in surface cultures.

5. A process according to claim 1, wherein the aqueous nutrient medium is composed of about 2.6% of de-oiled soybean meal,

1.0% of dextrin,

1.0% of glucose in the form of sucrose,

0.3% of magnesium sulfate heptahydrate, and 0.2% of calcium carbonate.

6. A process according to claim 1, wherein the aqueous solution of a volatile weak base used for elution is an aqueous solution of pyridine.

7. A process according to claim 1, wherein the aqueous solution of a volatile weak base used for elution is an aqueous solution of ammonia.

8. A process according to claim 4, wherein a lower alkanol is added to the concentrated eluate, the precipitate is dissolved in water, a lower alkanol is added to said aqueous solution, and the precipitated crystals of O-carbamyl-D-serine are removed from the mother liquor.

References Cited in the file of this patent UNITED STATES PATENTS 2,773,878 Shull et a1 Dec. 11, 1956 2,789,983 Harned Apr. 23, 1957 FOREIGN PATENTS 729,208 Great Britain May 4, 1955 OTHER REFERENCES Nature, vol. 173, pp. 71 to 73 (1954). 

1. IN A PROCESS OF PRODUCING O-CARBAMYL-D-SERINE, THE STEPS COMPRISING CULTIVATING STREPTOMYCES POLYHROMOGENUS IN AN AQUEOUS, NUTRIENT MEDIUM CONTAINING PROTEINS, CARBOHYDRATES, AND MINERAL SALTS AND HAVING A PH BETWEEN ABOUT 6.0 AND ABOUT 5.5 UNDER AEROBIC CONDITIONS AND WITH AGITATION AT A TEMPERATURE BETWEEN ABOUT 20* C. AND ABOUT 32* C. UNTIL FORMATION OF O-CARBAMYL-DSERINE IS COMPLETED, AND RECOVERING THE RESULTING O-CARBAMYL-D-SERINE FROM THE NUTRIENT MEDIUM BY ADSORPTION TO A SULFONIC ION EXCHANGE RESIN IN ACID FORM AND SUBSEQUENT ELUTION BY MEANS OF AN AQUEOUS SOLUTION OF A VOLATILE WEAK BASE SELECTED FROM THE GROUP CONSISTING OF PYRIDINE AND AMMONIA. 